Snail, new tumoral progression marker and target protein of new antitumoral compounds

ABSTRACT

The Snail transcription factor has been identified as a repressor of the expression of E-cadherin. The expression of Snail induces invasive and metastatic capacity in tumor cells. This invention presents: a new target protein, Snail, for the identification of new antitumoral compounds and a new diagnostic tumor marker, indicative of tissue invasion and metastatic capacity.

This is a 371 of PCT/ES00/00226 filed Jun. 27, 2000.

INVENTION SECTOR

Biomedicine. Target protein for antitumoral compounds.

System for identification of candidates for antitumoral compounds.

Tumour invasion and metastasis markers, their use as diagnostic markersof the disease and as a guide to medical professionals in the selectionor evaluation of treatments.

PRIOR ART

The protein E-cadherin has not only been shown to mediate intercellularadhesion of epithelial cells during embryonic development and in adulttissues, but also to be implicated in the phenotypic transformationobserved in epithelial tumours during their progression into invasivetumours. In this process of invasion by the tumour cells, expression ofthe protein E-cadherin is reduced or abolished and this loss isassociated with the acquisition of migratory properties. Functionalalterations of E-cadherin and/or its associated proteins, catenins, havebeen associated with de-differentiation and greater aggressivity oftumours (Takeichi, M. Cadherins in cancer: implications for invasion andmetastasis. Curr. Op. Cell Biol. 5, 806-811 (1993); Birchmeier, W. &Behrens, J. Cadherin expression in carcinomas: role in the formation ofcell junctions and the prevention of invasiveness. Biochim. Biophys.Acta 1198, 11-26 (1994)] and have even been seen to be implicated in thetransition from adenomas to invasive carcinomas [Perl, A. K., P.Wilgenbus, U. Dahl, H. Semb & Christofori, G. A causal role forE-cadherin in the transition from adenoma to carcinoma. Nature 392,190-193 (1998).]. For all of these reasons, the E-cadherin gene has beenconsidered to be a tumoral invasion suppressor gene [Frixen, U. H., etal. E-cadherin-mediated cell-cell adhesion prevents invasiveness ofhuman carcinoma cells. J. Cell Biol 113, 173-185 (1991); Vleminckx, K.,Vakaet, L. J., Mareel, M., Fiers, W. & Van Roy, F. Genetic manipulationof E-cadherin expression by epithelial tumor cells reveals an invasionsuppressor role. Cell 66, 107-119 (1991); Miyaki, M. et al. Increasedcell-substratum adhesion, and decreased gelatinase secretion and cellgrowth, induced by E-cadherin transfection of human colon carcinomacells. Oncogene 11, 2547-2552 (1995); Llorens, A. et al. Downregulationof E-cadherin in mouse skin carcinoma cells enhances a migratory andinvasive phenotype linked to matrix metalloproteinase-9 gelatinaseexpression. Lab. Invest. 78, 1-12 (1998).] so that the molecularmechanisms which control its expression or function are of the utmostimportance in increasing our knowledge of tumour invasion processes.

Expression of the E-cadherin gene is regulated by several elementslocated in the 5′ proximal region of its promoter [Behrens, J., Löwrick,O., Klein, H. L. & Birchmeier, W. The E-cadherin promoter: functionalanalysis of a GC-rich region and an epithelial cell-specific palindromicregulatory element. Proc. Natl. Acad. Sci. USA 88, 11495-11499 (1991);Ringwald, M., Baribault, H., Schmidt, C. & Kemler, R. The structure ofthe gene coding for the mouse cell adhesion molecule uvomorulin. NucleicAcids Res. 19, 6533-6539 (1991); Bussemakers, M. J., Giroldi, L. A., vanBokhoven A. & Schalken, J. A. Transcriptional regulation of the humanE-cadherin gene in human prostate cancer cell lines: characterization ofthe human E-cadherin gene promoter. Biochem. Biophys. Res. Commun. 203,1284-1290 (1994); Giroldi, L. A. et al. Role of E-boxes in therepression of E-cadherin expression. Biochem. Biophys. Res. Commun. 241,453-458 (1997)]. Of these, the E-pal element, which contains twoE-boxes, has been identified in the E-cadherin promoter in mice (betweenpositions −90 and −70) and is important because it acts as a repressorin normal cells and transformed cells deficient in E-cadherin [Behrens,J., Löwrick, O., Klein, H. L. & Birchmeier, W. The E-cadherin promoter:functional analysis of a GC-rich region and an epithelial cell-specificpalindromic regulatory element. Proc. Natl. Acad. Sci. USA 88,11495-11499 (1991); Hennig, G., Lowrick, O., Birchmeier, W & Behrens, J.Mechanisms identified in the transcriptional control of epithelial geneexpression. J. Biol. Chem. 271, 595-602 (1996); Faraldo; M. L., Rodrigo,I., Behrens, J., Birchmeier, W & Cano, A. Analysis of the E-cadherin andP-cadherin promoters in murine keratinocyte cell lines from differentstages of mouse skin carcinogenesis. Mol. Carcinog. 20, 33-47 (1997);Rodrigo, I., Cato, A. C. B. & Cano, A. Regulation of E-cadherin geneexpression during tumor progression: the role of a new Ets-binding siteand the E-pal element. Exp. Cell Res. 248, 358-371 (1999)]. Thetranscription factors which interact with this element or in thecorresponding region of the promoter of the human cadherin gene[Bussemakers, M. J., Giroldi, L. A., van Bokhoven A. & Schalken, J. A.Transcriptional regulation of the human E-cadherin gene in humanprostate cancer cell lines: characterization of the human E-cadheringene promoter. Biochem. Biophys. Res. Commun. 203, 1284-1290 (1994);Giroldi, L. A. et al. Role of E-boxes in the repression of E-cadherinexpression. Biochem. Biophys. Res. Commun. 241, 453-458 (1997)] areunknown.

Potential transcription factors which are repressors of the expressionof the E-cadherin gene could be of great value in the identification ofnew antitumoral candidates which act by inhibiting the function of thesefactors, and consequently the invasive and metastatic process.Furthermore, their presence could be used as markers of tumour spreadand malignancy.

DESCRIPTION Brief Description

The transcription factor Snail has been identified as a repressor of theexpression of E-cadherin, as it has a direct interaction with the E2 boxof the E-pal element of the promoter. The ectopic expression of Snail inepithelial cells induces epithelial-mesenchymal transition and theacquisition of migratory properties concomitant with inhibition ofE-cadherin expression and the loss of other epithelial differentiationmarkers. This invention presents and includes the following:

a new target protein for the identification of new antitumoralcompounds, and

a new marker of tumour invasion and metastasis and its application as adiagnostic marker of the disease and as a guide to medical professionalsin the selection or evaluation of treatments.

Detailed Description

Snail is a Transcription Factor Which Acts as a Direct Repressor ofE-cadherin Expression.

Identification of transcription factors which interact with the E-palelement was undertaken by means of a one-hybrid approximation using themouse E-pal sequence (−90/−70) oligomerised to direct the expression ofthe HIS3 gene of S. cerevisiae as bait and a cDNA gene library of NIH3T3fused to the GAL4 activation domain as a prey. A total of 130 cloneswere isolated, capable of interacting with (and directing thetranscription of the reporter gene HIS3) the construction containing thenative E-pal element; they did not recognise the mutated oligomericelement. This mutated form of the E-pal element contains 2 modifiedbases (TT instead of GC) which eliminate the E2 box. This mutated formhas been described as being responsible for abolishing the repressoreffect in the E-cadherin Promoter in mice (Hennig, G., Löwrick, O.,Birchmeier, W. & Behrens, J. Mechanisms identified in thetranscriptional control of epithelial gene expression. J. Biol. Chem.271, 595-602 (1996); Faraldo, M. L., Rodrigo, I., Behrens, J.,Birchmeier, W & Cano, A. Analysis of the E-cadherin and P-cadherinpromoters in murine keratinocyte cell lines from different stages ofmouse skin carcinogenesis. Mol. Carcinog. 20, 33-47 (1997).

Sequentiation of the isolated clones revealed that 49% of them containedinserts which encoded the complete or partial sequence of the mouseSnail cDNA [Nieto, M. A., Bennet, M. F., Sargent, M. G. & Wilkinson, D.G. Cloning and developmental expression of Sna, a murine homologue ofthe Drosophila snail gene. Development 116, 227-237 (1992); Smith, D.E., Del Amo, F. F. & Gridley, T. Isolation of Sna, a mouse homologous tothe Drosophila gene snail and escargot: its expression pattern suggestsmultiple roles during postimplantation development. Development 116,1033-1039 (1992)], while one single clone encoded a partial sequence ofthe mouse Slug cDNA.

To determine the effect of Snail as a transcription factor in thecontext of the proximal region of the E-cadherin promoter (−178/+92),the complete sequence of Snail DNA was subcloned in an expression vector(pcDNA3) and its activity was analysed by cotransfection in two mouseepidermal keratinocyte cell lines, MCA3D and PDV. Both lines hadpreviously been characterised as having a high level of E-cadherinexpression and promoter activity [Faraldo, M. L., Rodrigo, I., Behrens,J., Birchmeier, W & Cano, A. Analysis of the E-cadherin and P-cadherinpromoters in murine keratinocyte cell lines from different stages ofmouse skin carcinogenesis. Mol. Carcinog. 20, 33-47 (1997); Rodrigo,

I., Cato, A. C. B. & Cano, A. Regulation of E-cadherin gene expressionduring tumor progression: the role of a new Ets-binding site and theE-pal element. Exp. Cell Res. 248, 358-371 (1999); Navarro, P. et al. Arole for the E-cadherin cell-cell adhesion molecule in tumor progressionof mouse epidermal carcinogenesis. J. Cell Biol. 115, 517-533 (1991)].Cotransfection of Snail in MCA3D cells(FIG. 1A) and PDV (FIG. 1B)produced strong repression of the native E-cadherin promoter (95% and75%, respectively), but not of the promoter containing the mutated E2box (FIG. 1). These results confirm those obtained by the one-hybridscreening method and demonstrate that Snail is a direct repressor of thetranscription of the E-cadherin gene acting through its binding to theE2 box of the E-pal element.

Snail Induces the Fibroblastic Conversion of Epithelial Cells and theAcquisition of Migratory Properties.

To obtain more information about the role of the Snail protein in theregulation of the E-cadherin gene and its participation inepithelial-mesenchymal transition, it was ectopically expressed inseveral cell lines. Transient expression of Snail was initially analysedin the keratinocyte lines MCA3D and PDV, which offer the advantage ofbeing able to grow in isolated groups, establishing strong intercellularcontacts mediated by E-cadherin even at low density, and exhibit verylow cell motility [Navarro, P. et al. A role for the E-cadherincell-cell adhesion molecule in tumor progression of mouse epidermalcarcinogenesis. J. Cell Biol. 115, 517-533 (1991); Lozano, E & Cano, A.Cadherin/catenin complexes in murine epidermal keratinocytes: E-cadherincomplexes containing either b-catenin or plakoglobin contribute tostable cell-cell contacts. Cell Adh. Commun. 6, 51-67 (1998); Gómez M.,Navarro P. & Cano A. Cell adhesion and tumor progression in mouse skincarcinogenesis: increased synthesis and organization of fibronectin isassociated with the undifferentiated spindle phenotype. Invasion &Metastasis 14, 17-26 (1994); Frontelo, P. et al. Transforming growthfactor b1 induces squamous carcinoma cell variants with increasedmetastatic abilities and a disorganized cystoskeleton. Exp. Cell Res.244, 420-432 (1998)]. The overexpression of Snail eliminatedintercellular contacts within the 24-28 hours after transfection in bothcell types as a consequence of inhibition of the expression ofE-cadherin (FIGS. 2b and 2 f) and other associated proteins, such asplakoglobin. Simultaneously with these changes, the morphology of thecells transfected with Snail was greatly altered. Abundant membranousprolongations and long filaments resembling filopodia were observed inboth cell lines.

Stable expression of Snail was carried out in the cell line MDCK, whichexhibits a “prototypical” epithelial phenotype and appears as amonolayer in culture. This phenotype does not seem to be affected byexpression of the control vector in six independent isolated clones(FIG. 3a), which maintain the expression of E-cadherin (FIG. 3b) andplakoglobin (FIG. 3c) in intercellular contacts. However, stableexpression of Snail induces a dramatic conversion to a completelyundifferentiated fibroblastic phenotype. MDCK cells transfected withSnail lose the ability to grow as a monolayer and to inhibit by contact.Instead, these cells form overlying networks with extremely longmembranous extensions (FIG. 3d). Analysis of E-cadherin and plakoglobinexpression showed the loss of both molecules in MDCK cells transfectedwith Snail (FIGS. 3e and f). Additionally, stable expression of Snail inMDCK cells induced a strong migratory behaviour, which was demonstratedby tests of wound-healing in cultures (FIG. 4).

Snail is Expressed in Undifferentiated Tumours and in Invasive Regionsof Epidermoid Carcinomas.

Analysis of the endogenous expression of Snail by RT-PCR in a panel ofcell lines with varying E-cadherin expression demonstrated an inversecorrelation between the expression of both molecules and a relationshipbetween the expression of Snail an their invasive and metastaticcapacity (FIG. 5). E-cadherin was observed in the epithelial,non-tumoral MCA3D cell line an in the PDV tumoral cell line, which inspite of its tumoral origin showed no invasive or metastatic capacity.However, the presence of Snail was not found in any of the cell lines.In contrast, in the tumour cell lines with invasive and metastaticcapacity, HaCa4 and CarB, the absence of E-cadherin is associated withthe presence of Snail.

Expression of Snail was analysed by in situ hybridisation in epidermoidtumours induced in immunodepressed mice, either by different cell linesor by chemical treatment of the mouse's skin. In both cases, highexpression of Snail was observed in undifferentiated tumours (FIGS. 6fand h) and in zones of invasion in epidermoid carcinomas (FIG. 6l) whichhad lost expression of E-cadherin (FIG. 6k). In contrast, no expressionof Snail was detected in non-invasive, well-differentiated tumours(FIGS. 6b and 6 d).

Taken together, these data demonstrate that Snail is a direct repressorof E-cadherin expression, implicated in the epithelial-mesenchymaltransition which occurs during tumour invasion. The presence of Snail,therefore, is a new marker of tumour progression, specificallyassociated with the acquisition of the invasive and metastaticphenotype, which allows it to be used as a diagnostic marker of tumoursin humans and as a guide to medical professionals in the selection andevaluation of antitumoral treatments and forms part of this invention.

Moreover, these data clearly indicate the direct inductive role of Snailin the acquisition of these characteristics of tumour invasion andmetastasis, so that Snail may be considered a target protein for newantitumoral compounds. Experiments to identify new antitumoralcandidates can be developed from this protein, based on cell linestransformed by the Snail protein, in which analysis of the regulation ofSnail expression by an antitumoral candidate would identify newantitumoral compounds, which form part of this invention. Analysis ofthe regulation of Snail expression could be carried out by determiningthe presence or absence of Snail after contact with the antitumoralcandidate, or by means of another type of signal inhibiting the Snailfunction in cells transformed with reporter genes, eg HIS3 and LacZ,which form part of this invention.

DESCRIPTION OF THE FIGURES

FIG. 1.—Snail represses the activity of the E-cadherin promoter inepithelial cell lines. MCA3D (FIG. 1A) and PDV cells (FIG. 1B) weretransfected with the native E-cadherin promoter (wt-178) or with amutated version (mE-pal) fused to the gene marker CAT in the presence of1 μg of control vector pcDNA3 or containing Snail. The graph shows thelevels of CAT activity of the promoter. Promoter activity is expressedas a measurement relative to that of the native promoter in the presenceof the control vector.

FIG. 2.—Transient expression of Snail in epidermal keratinocytes inducesloss of E-cadherin and plakoglobin and loss of cell-cell adhesion. MCA3D(a-d) and PDV (e-h) cells were transfected with control vector (mock,a,c,e,g) or that containing Snail cDNA (b,d,g,h) and the presence ofE-cadherin and plakoglobin was analysed by immunocytochemistryvisualised by confocal microscopy after 48 hours.

FIG. 3.—Stable transfection of Snail in MDCK epithelial cells inducesepithelial-mesenchymal conversion concomitant with the loss ofE-cadherin and plakoglobin. Phase contrast images of cells transfectedwith the control vector (a) and with the vector containing Snail (d).Confocal microscopy images which show expression of E-cadherin (b,e) andplakoglobin (c,f) in control and transfected Snail cells, respectively.

FIG. 4.—Snail induces a migratory phenotype in epithelial cells. Themigratory behaviour was analysed in an in vitro wound model. MDCKcontrol cultures (mock) or cultures transfected with Snail werescratched with the tip of a pipette and photographs were takenimmediately (t=0, a,d) and 10 hours later (b,e).

FIG. 5.—Analysis of the endogenous expression of Snail by RT-PCR in apanel of cell lines. The endogenous expression of Snail is inverselycorrelated with that of E-cadherin in normal and transformed mousekeratinocyte cell lines.

FIG. 6.—Snail expression is associated with invasive carcinomas andinvasive areas in epidermoid carcinomas. Tumours were induced in nudemice with PDV (a-d) or CarB (e-h) cells or by chemical treatment (i-l).They were analysed by hybridisation in situ with E-cadherin(a,c,e,g,i,k) or Snail (b,d,f,h,j,l) probes. E-cadherin is expressed indifferentiated areas of epidermoid carcinomas (a,c,i,k), while there isno expression of Snail in these tumours (b,d,j). Invasive carcinomas donot express E-cadherin (e,g) but do express Snail (f,h). In chemicallyinduced tumours, Snail expression is observed in undifferentiatedinvasive areas (l), which do not express E-cadherin (k).

EXAMPLES Example 1 Isolation of Snail cDNA in Mice Using the One-hybridTechnique.

The oligonucleotide which contains the sequence of the E-pal element ofthe mouse E-cadherin promoter (CD-E) (nucleotides −90 to −70) containingtargets for the restriction enzymes SalI in 5′ and XhoI in 3′ wasligated in direct sense for a total of 6 complete repetitions usingconventional techniques, isolation in polyacrylamide gels and cloning inpHISi vector (Clontech, Palo Alt, Calif.) which contains the reportergene HIS3 of S. cerevisia and replication elements of yeast, bacteriaand appropriate selection genes. In this way, the expression of the HIS3gene remains under the control of the multimerised E-pal element.Correct insertion of the regulatory sequences was verified by PCR,digestion with appropriate restriction enzymes and sequentiation. Thebait vector thus generated was denominated pHIS-E6. The same method wasused to generate vectors into which a mutant version of the E-palelement was introduced, also ligated 6 times in direct sense, in whichthe two central oligonucleotides, GC, were replaced by TT. The mutantbait vector generated was denominated pHIS-mE6. The bait vectors pHIS-E6and pHIS-mE6 were independently integrated in the chromosomal locus UPA3of the yeast strain YM4271 (Clontech, Palo Alto, Calif.) by the usualtechniques for transformation and selection of stable strains whichmaintain growth in the presence of 20 mM 3-aminotriazole (3AZT). Thestrains selected were denominated E-pal HIS3 (native E-pal construct)and mE-pal HIS3 (mutated E-pal construct). The yeast strain E-pal HIS3was subjected to transformation with a commercial gene library of cDNAfron NIH3T3 cells which contains different inserts of cDNA fused to theGAL4 activation domain in the pACT2 vector (Clontec Palo Alto, Calif.),previously amplified to obtain a titre of 3×10⁶ independent clones usingconventional techniques. Transformant yeasts were selected for theirability to grow in the absence of Histidine and in the presence of 20 mM3ATZ, and 300 independent clones were isolated. The plasmids containingthe different sequences of cDNA were isolated from the transformantyeasts and were later used to transform E. coli (DH5a strain),recovering 221 independent E. coli clones, from which the correspondingplasmids were isolated. To eliminate false positives, the 221 plasmidswere independently introduced in parallel into the previously generatedyeast strains containing the HIS3 gene under the control of the wildE-pal element (E-pal HIS3 strain) or mutated E-pal (mE-pal HIS3 strain),selecting those plasmids which conferred growth in the absence ofhistidine and leucine and in the presence of 20 mM 3ATZ exclusively inthe strain E-pal His3; the total number selected was 130. Inserts ofthese plasmids were initially analyse using digestion with variousrestriction enzymes and sequenced in an automatic sequencer. Thesequences obtained were analysed in cDNA databanks using the BLAST/FASTAprogramme. 49% of the clones identified encoded the total or partialmouse Snail cDNA sequence.

Example 2 Transitory Stable Transfection of mSnail in Epithelial Cells.

The complete mouse Snail cDNA sequence (mSnail) contained in one of theclones identified in the one-hybrid screening was isolated from thepACT2 vector by digestion with the restriction enzymes EcoRIy HindIIIand subcloned in the EcoRI/HindIII sites of the pcDNA3 vector(Invitrogene), which contains the neo gene conferring resistance to theantibiotic G418 and was sequenced at both ends. The vector thusgenerated was denominated pcDNA3-mSnail.

The mouse E-cadherin promoter construct, −178, which contains the genesequences −178 to +92 pb fusioned to the CAT reporter gene(Chloramphenicol Acetyl Transferase), and the mEpal construct (in whichthe two central GC nucleotides of the E-pal element were mutated by TT)have been described previously [Behrens, J., Löwrick, O., Klein, H. L. &Birchmeier, W. The E-cadherin promoter: functional analysis of a GC-richregion and an epithelial cell-specific palindromic regulatory element.Proc. Natl. Acad. Sci. USA 88, 11495-11499 (1991); Hennig, G., Löwrick,O., Birchmeier, W & Behrens, J. Mechanisms identified in thetranscriptional control of epithelial gene expression. J. Biol. Chem.271, 595-602 (1996); Faraldo, M.L., Rodrigo, I., Behrens, J.,Birchmeier, W & Cano, A. Analysis of the E-cadherin and P-cadherinpromoters in murine keratinocyte cell lines from different stages ofmouse skin carcinogenesis. Mol. Carcinog. 20, 33-47 (1997)] and wereprovided by Dr J. Behrens.

a) Analysis of the Activity of the E-cadherin Promoter.

MCA3D AND PDV cells were seeded at subconfluence (3×10⁵ cells/6 cmdiameter plate, P-60) in HamF12 growth medium containing 10% foetalbovine serum (Gibco) and incubated for 24 hours at 37° C. in anincubator containing 5% CO₂ with a humidity of 95%. The medium was thenreplaced by fresh DMEM medium with 10% foetal bovine serum and kept inthe incubator for a further 6 hours. The cultures underwent transfectionusing Lipofectamin Plus (Life Technologies), following the supplier'sinstructions, using 2.5 μg of construct −178, mE-pal or the controlvector pCATbasic (with no promoter sequences) (Promega) and in thepresence of 1 μg of pcDNA3-mSnail or the empty pcDNA3 plasmid. As anadditional control, the activity of the E-cadherin promoter constructswas compared with that of the vector pCAT-control (Promega) containingthe CAT reporter gene under the control of the SV-40 promoter, and thusthe cells were transfected in parallel with this vector. Efficiency oftransfection was analysed by cotransfection in all the cultures with 2.5μg of the CMV-Luc plasmid which contains the luciferase reporter geneunder the control of the cytomegalovirus promoter. Twenty-four hoursafter transfection the medium was discarded and after washing with PBSthe cells were collected by gently scraping the plates and werecentrifuged (2000 rpm for 4 minutes). Extracts were obtained byresuspending the cellular pellet in 100 μl of a buffer containing 10mMphosphate pH 8.0 and subjected to 4 cycles of freezing in liquidN₂-thawing at 37° C. Luciferase activity was initially determined inaliquots of 5 μl using a commercial kit and a luminometer. Aliquots ofthe different extracts containing equivalent luciferase activities wereanalysed for CAT activity, using C¹⁴-chloramphenicol (Amersham) assubstrate and Acetyl-CoA (Sigma) as cofactor, following the methoddescribed previously [Faraldo, M. L., Rodrigo, I., Behrens, J.,Birchmeier, W & Cano, A. Analysis of the E-cadherin and P-cadherinpromoters in murine keratinocyte cell lines from different stages ofmouse skin carcinogenesis. Mol. Carcinog. 20, 33-47 (1997); Rodrigo, I.,Cato, A. C. B. & Cano, A. Regulation of E-cadherin gene expressionduring tumor progression: the role of a new Ets-binding site and theE-pal element. Exp. Cell Res. 248, 358-371 (1999)]. The CAT activityobtained in MCA3D and PDV cells for the −178 construct of the nativeE-cadherin promoter was 70% and 50%, respectively, of that of thepCAT-SV40 vector. The CAT activities obtained were normalised to thatobtained with the −178 construct in the presence of the empty pcDNA3vector in each cell line. Transfection assays were performed inequivalent duplicate cultures of each of the cell lines for all theexperimental conditions analysed.

b) The Effect of the Expression of mSnail in the Cell Phenotype and theExpression of Epithelial Markers.

Transient transfections were performed with 2 μg of mSnail(pcDNA3-mSnail vector) and “mock” controls (empty pcDNA3 vector) inmouse keratinocyte lines MCA3D and PDV, following the proceduredescribed previously, except that the cells were seeded on circularcoverslips (1.2 cm in diameter) placed on top of the P-60 plates. At 24and 48 hours after transfection, the glass plates corresponding to thetwo experimental conditions of each cell line were fixed with methanol(−20° C.) for 30 s, and were analysed for E-cadherin and plakoglobinexpression by immunofluorescence [Navarro, P. et al. A role for theE-cadherin cell-cell adhesion molecule in tumor progression of mouseepidermal carcinogenesis. J. Cell Biol. 115, 517-533 (1991)]. The imageswere analysed using a confocal microscope (Leica).

Stable transfections of mSnail and “mock” control were carried out onthe MDCK epithelial line, grown in DMEM medium, 10% foetal bovine serum,in parallel cultures and following the protocol described previously.Between 48 and 72 hours after transfection, when the cultures reachedconfluence, the medium was changed for fresh medium and 400 μg/ml ofG418 were added, selecting the cells resistant to G418 after 2-3 weeksof growth in the presence of the antibiotic. The total populationgenerated (the “pool”) in both types of culture (mSnail and mock) wascollected, and independent clones were obtained by dilution. To do this,100 cells of each type of population were seeded on P-100 plates (10 cmdiameter) and grown in DMEM medium, 10% foetal bovine serum and 400μg/ml of G418. Independent colonies resistant to G418 were obtainedafter a further 2-3 weeks, and these were isolated by trypsinisationusing cloning cylinders (internal diameter 5 mm) and amplified bysuccessive passages on culture plates of increasing size(T6->F12.5->F25->F75), maintaining antibiotic pressure in all phases ofthe culture. A total of 10 independent clones were isolated from themSnail transfection and 6 independent clones from the mock transfection.The different clones were analysed for E-cadherin and plakoglobinexpression by immunofluorescence (confocal microscope analysis) andimmunotransference, and for mSnail expression by RT-PCR after extractionof the RNA-polyA+of the different clones and the use of appropriateamplimers to amplify a fragment of 388 base pairs, in accordance withthe mSnail cDNA sequence.

Example 3 Obtaining Tumours

a) Tumours Induced in Immunodepressed Mice by Cell Lines.

Tumours were induced in 8-week-old athymic male nu/nu mice of the BalCstrain by subcutaneous injection of PDV, HaCa4 or CarB cells, asdescribed previously [Navarro, P. et al. A role for the E-cadherincell-cell adhesion molecule in tumor progression of mouse epidermalcarcinogenesis. J. Cell Biol. 115, 517-533 (1991)]. The different celllines grew to confluence in F75 bottles, and were trypsinised andresuspended in phosphate buffered saline (PBS) at a density of 1×10⁷cells/ml in PBS. The cells were subcutaneously injected into both flanksof each mouse (1×10⁶ cells/injection site) using insulin syringes and 25gauge hypodermic needles. Usually 3 animals were inoculated for eachcell line (6 injection sites/line). The animals were obtained from theIFA-CREDO production unit (France) and were kept in sterile conditionsin the installation specifically intended for such animals in the animalhouse of the Instituto de Investigaciones Biomédicas (IIB) in accordancewith the centre's regulations for animal handling. The injected animalswere observed 3 times a week; the appearance of tumours was determinedby visual inspection and measurement of their size was done by caliper.The animals were sacrificed by asphyxia in ether when the externaldiameter of the tumours reached 1.5-2.0 cm. The tumours were removed, afraction was fixed in formaldehyde for subsequent histological analysis,and the remainder was immediately frozen in isopentane directly cooledin a bath of liquid nitrogen or embedded in OCT (Tissue Tek). Thesamples were stored at −70° C. for later use.

b) Induction of Tumours in Mouse Skin by Chemical Carcinogenesis.

Tumours were induced in the dorsal skin of 8-10-week-old BalC miceobtained from the production unit of the IIB animal house, using thetwo-stage DMBA/TPA protocol as described [Cano, A. et al. Expressionpattern of the cell adhesion molecules E-cadherin, P-cadherin andintegrin is altered in pre-malignant skin tumors of p53-deficient mice.Int. J. Cancer 65, 254-262 (1996)]. One week before the start of theexperiment, the backs of the animals (20 in total) were shaved, afterwhich a single dose of the carcinogen dimethylben(z)anthracene (DMBA) ata concentration of 50 μg/ml dissolved in acetone was applied. A weeklater, the promotion was begun by topical application of phorboltetradecanoylphorbolacetate at a concentration of 50 μg/ml dissolved inethanol. The TPA was applied every 3 days for a total of 30 weeks. Theanimals were kept under weekly observation for a total of 50 weeks. Atthe end of 10 weeks of treatment with TPA the appearance of the firstpapilloma-type tumours was detected, and these continued to appearthroughout the treatment and subsequently, at an average rate of, 5-6tumours per mouse. A small proportion of the papillomas (5%) progressedto carcinomas after the end of the TPA treatment. The animals weresacrificed at different intervals and the tumours were extracted andfrozen as previously described.

What is claimed is:
 1. A method for determining the invasive andmetastatic capacity of an epithelial tumour, said method comprising: a.obtaining a biological sample from said epithelial tumour; b.determining whether a diagnostic marker, Snail, is present in saidbiological sample; and c. comparing said diagnostic marker determined tobe present in said biological sample with its absence in a controlsample, the presence of said marker in said biological sample beingindicative of the invasive and metastatic capacity of said epithelialtumour.
 2. The method according to claim 1, wherein said step ofdetermining the presence of said diagnostic marker, Snail, is carriedout by using specific anti-Snail antibodies generated from Snailprotein.
 3. The method according to claim 1, wherein said step ofdetermining the presence of said diagnostic marker, Snail, is carriedout by in situ hybridization for a genetic precursor of said diagnosticmarker.
 4. The method according to claim 1, wherein the step ofdetermining the presence of said diagnostic marker, Snail, is carriedout by RT-PCR for a genetic precursor of said diagnostic marker, basedon extraction of RNA polyA+of tumour samples and control tissue and theamplification of encoding sequences for said diagnostic marker usingappropriate amplimer.